Fluorescence microscopy presentation

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1. Fluorescence microscopy Mandira S. Bhosale MSc part 1 sem 2 FMBCH2122005

2. History: Fluorescence was first discovered in 1845 by Fredrick W. Herschel.

3. History: British scientist Sir George G. Stokes further studied and discovered, that fluorescence emission from an object represents a longer wavelength than the UV light that originally excited the object . He coined the term Fluorescence. Hence , the shift from shorter wavelength to the higher wavelength known as Stokes shift.

4. History : I n the early 1900s, the first uses of fluorophores in biological investigations were performed to stain tissues, bacteria, and other pathogens. This was later developed into fluorescence microscopy by Carl Zeisi and Carl Reichert. Fluorescence labeling was achieved by Ellinger and Hirt in the early 1940s. The cloning of green fluorescent protein (GFP) was achieved in the early 1990s first and was easily applied to fluorescence microscopy.

5. Fluorescence microscopy: Fluorescence : Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation . + Microscopy : Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye.

6. Fluorescence microscopy is study in which microbes or molecules observed under microscope with the help of fluorescence. A   fluorescence microscope  is an  optical microscope  that uses  fluorescence  instead of, or in addition to,  scattering ,  reflection , and  attenuation  or  absorption , to study the properties of organic or  inorganic  substances . Fluorescence microscopy uses fluorescence to examine the structural organization, spatial distribution of samples. It is particularly used to study samples that are complex and cannot be examined under conventional transmitted-light microscope .

7. Fluorescence microscope Vs light microscope: Light microscopes use light in the 400-700nm range , the range through which light is visible to the human eye ,but fluorescence microscopy uses much higher intensity light . light microscopy uses visible light, the resolution is more limited. Fluorescence microscopy , uses light produced by the fluorophores in the sample. light microscopes do still have a prominent place in microscopy however, both in combination with dyes and sometimes without. Fluorescence microscopy is used in conjunction with other kinds of light microscopy because it creates images from reflected light, rather than direct light.

8. Principle: Most cellular components are colorless and hence cannot be visualize under a normal light microscope. Hence they are stained with fluorescent dye , also known as fluorophores or fluorochromes. These molecules stains that components and absorbs the excited light of shorter wavelength and emits the light of longer wavelength. This emitted light is called as fluorescent light. Emitted fluorescent light produces an image of desired part of molecule and which then projected on computer screen. Or can be observed on microscope with naked eyes.

9. Components:

10. Light source : Four main types of light sources are used, including xenon arc lamps or mercury-vapor lamps with an excitation filter, lasers, and high- power LEDs. Lasers are mostly used for complex fluorescence microscopy techniques, while xenon lamps, and mercury lamps, and LEDs are commonly used for wide-field & epifluorescence microscopes.

11. Dichroic mirror: A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors. Excitation filter and emission filter: The exciter is a bandpass filter that passes only the wavelengths absorbed by the fluorophore, thus minimizing the excitation of other sources of fluorescence and blocking excitation light in the fluorescence emission band.  The emitter is typically a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band – especially the excitation light.

12. Detector : Detector analyze the emitted light it has electron imaging sensor and projects the final image of specimen on computer screen. Darkfield condenser: It provides a black background against which the fluorescent objects glow.

13. Preparation of specimen : Fluorescent dyes Tagging of proteins Immunofluorescence

14. Light shorter wavelength of lights (UV rays or blue light) generated from mercury vapor arc lamp passes through the excitation filter Which allows only the short wavelength of light to pass through and removes all other non-specific wavelengths of light. The filtered light is reflected by the dichroic filter and falls on the sample (Fluorophore-labeled). The fluorochrome absorbs shorter wavelength rays and emits rays of longer wavelength (lower energy) that passes through the emission filter. The emission filter blocks (suppresses) any residual excitation light and passes the desired longer emission wavelengths to the detector. Thus the microscope forms glowing images of the fluorochrome -labeled microorganisms against a dark background.

15. Examples: Fluorescent image of human stem cells stained with monoclonal antibodies markers under the microscopy nuclei in blue and actin microfilaments in green English daisy flower ,cross section through a flower head, fluorescence microscope image Blue stained DNA.

16. Types: Epifluorescence microscopes : The most common type of fluorescence microscope in which, excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective ). Confocal microscope: In this type of fluorescence microscope, high‐resolution imaging of thick specimens (without physical sectioning) can be analyzed using fluorescent-labeled dye .

17. Multiphoton microscope : In this type of microscope, multiphoton fluorescence excitation results in the capture of high-resolution three-dimensional images of specimen tagged with highly specific fluorophores . Total internal reflection fluorescence (TIRF) microscope : Total internal reflection fluorescence microscopy (TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices.

18. Advances in microscopy

19. Widefield microscopy Lightsheet microscopy Stochastic optical reconstruction microscopy Electron cryo microscopy

20. Fluorescence recovery after photobleaching (FRAP) Fluorescence correlation spectroscopy (FCS) Super resolution stimulated emission depletion (STED) Photoactivated localization microscopy (PALM) Structured illumination microscopy (SIM) combined with TIRF Deep imaging via emission recovery (DIVER) Nanoscale precise imaging by rapid beam oscillation (nSPIRO)

21. On 8 October 2014 , the Nobel prize in chemistry was awarded to Eric Betzig, William Moerner and Stefan Hell for the development of super resolved fluorescence microscopy

22. A dvantages: Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in live-cell imaging. Isolation of individual proteins with a high degree of specificity can be visualized. Different molecules can be stained with different colors, allowing multiple types of the molecule to be tracked simultaneously. These factors combine to give fluorescence microscopy a clear advantage over other optical imaging techniques, for both in vitro and in vivo imaging.

23. Limitations: Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching .. Cells are susceptible to phototoxicity, particularly with short-wavelength light. Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows observation of the specific structures which have been labeled for fluorescence.

24. Applications: Fluorescence Microscopy used for Cell Labeling Protein Characterization achieved by Fluorescence Microscopy. Fluorescence Microscopy in forensic field for latent fingerprints, GSR. Detection of molecular interaction e.g . Proteins, DNA Conformational changes , structural studies of molecules. Tissue characterization by auto fluorescence. Visualization of molecules with immunofluorescence. Characterization of new materials.

25. References: https://microscopeinternational.com/fluorescence-microscopy / https ://microbeonline.com/fluorescence-microscope-principle-types-applications / https://pubmed.ncbi.nlm.nih.gov/23585290 / https://allmymedicine.com/health-news/the-history-of-fluorescence-microscopy / http:// www.scholarpedia.org/article/Fluorescent_proteins https://microbenotes.com/fluorescence-microscope-principle-instrumentation-applications-advantages-limitations / https://microscopeinternational.com/widefield-fluorescence-microscopy / https://microscopeinternational.com/total-internal-reflection-fluorescence-microscopy / https://www.nobelprize.org/educational_games/physics/microscopes/fluorescence / https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835776 / https:// onlinelibrary.wiley.com/doi/10.1002/cyto.a.22029 https:// di.uq.edu.au/community-and-alumni/sparq-ed/cell-and-molecular-biology-experiences/immunofluorescence/background-immunofluorescence

26. Thank you!!